Lipophilic dye-based FRET assays for clostridial toxin activity

Abstract

Compositions useful for detecting Clostridial toxin activity comprising a cell that comprises a membrane-associated Clostridial toxin substrate comprising a first member of a fluorescence resonance energy transfer pair; and a Clostridial toxin recognition sequence including a cleavage site; and a membrane-associated second member of the FRET pair and methods useful for determining Clostridial toxin activity using such Clostridial toxin substrates.

What is claimed: 
     
       1. A cell comprising:
 a) a membrane-associated Clostridial toxin substrate, said substrate comprising
 i) a first member of a fluorescence resonance energy transfer (FRET) pair; 
 ii) a Clostridial toxin recognition sequence including a Clostridial toxin cleavage site; and 
 iii) a membrane targeting motif comprising Gln-Pro-Xaa-Arg-Val (SEQ ID NO:135) or Gln-Pro-Xaa-Arg-Ile (SEQ ID NO:136); 
 wherein the Clostridial toxin recognition sequence intervenes between the first FRET pair member and the membrane targeting domain; 
 
 b) a membrane-associated second member of a FRET pair; and 
 c) a receptor that binds a Clostridial toxin; 
 wherein the cell is capable of Clostridial toxin intoxication; 
 wherein the FRET pair comprises an acceptor having an absorbance spectrum overlapping the emission spectrum of a donor fluorophore; and wherein under the appropriate conditions, resonance energy transfer is exhibited between the acceptor and the donor fluorophore. 
 
     
     
       2. The cell of  claim 1 , wherein the Clostridial toxin substrate is expressed from a nucleic acid molecule. 
     
     
       3. The cell of  claim 1 , wherein the cell is a neuronal cell. 
     
     
       4. The cell of  claim 1 , wherein the cell is a non-neuronal cell. 
     
     
       5. The cell of  claim 1 , wherein the first FRET pair member is a fluorescent protein, a fluorophore binding protein, or a fluorescent dye. 
     
     
       6. The cell of  claim 1 , wherein the second FRET pair member is a lipophilic dye. 
     
     
       7. The cell of  claim 1 , wherein the first FRET pair member is the acceptor and the second FRET pair member is the donor fluorophore. 
     
     
       8. The cell of  claim 1 , wherein the first FRET pair member is the donor fluorophore and the second FRET pair member is the acceptor. 
     
     
       9. The cell according to  claim 1 , wherein the Clostridial toxin recognition sequence comprises a BoNT/A recognition sequence including a BoNT/A cleavage site, a BoNT/B recognition sequence including a BoNT/B cleavage site, a BoNT/C1 recognition sequence including a BoNT/C1 cleavage site, a BoNT/D recognition sequence including a BoNT/D cleavage site, a BoNT/E recognition sequence including a BoNT/E cleavage site, a BoNT/F recognition sequence including a BoNT/F cleavage site, a BoNT/G recognition sequence including a BoNT/G cleavage site, or a TeNT recognition sequence including a TeNT cleavage site. 
     
     
       10. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of SNAP-25, the six consecutive residues comprising Gln-Arg. 
     
     
       11. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of VAMP, the six consecutive residues comprising Gln-Phe. 
     
     
       12. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of SNAP-25, the six consecutive residues comprising Arg-Ala. 
     
     
       13. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of Syntaxin, the six consecutive residues comprising Lys-Ala. 
     
     
       14. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of VAMP, the six consecutive residues comprising Lys-Leu. 
     
     
       15. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of SNAP-25, the six consecutive residues comprising Arg-Ile. 
     
     
       16. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of VAMP, the six consecutive residues comprising Gln-Lys. 
     
     
       17. The cell of  claim 1 , wherein the Clostridial toxin recognition sequence comprises at least six consecutive residues of VAMP, the six consecutive residues comprising Ala-Ala. 
     
     
       18. The cell of  claim 1 , wherein the receptor is an endogenous Clostridial toxin receptor. 
     
     
       19. The cell of  claim 1 , wherein the receptor is an exogenous Clostridial toxin receptor.